As early as 1971, a so-called “suppressor” cell population was first described by Gershon and Kondo when they transferred antigen-specific tolerance to naïve animals by transferring antigen-experienced T cells (Gershon and Kondo, Immunology; 21: 903-914 (1971)). Due to conflicting results the concept of T cell suppression however fell into oblivion in the late 1980s.
Sakaguchi et al. were the first to describe now termed “regulatory” T cells (Treg cells) by identifying a population of CD4+ T cells highly expressing CD25 and preventing autoimmunity in a murine model (Sakaguchi et al., J. Immunol. 155: 1151-1164 (1995)). In the following years a number of reports enlightened major aspects of Treg cell biology, characterizing different T cell subpopulations with regulatory properties including naturally occurring CD4+CD25high Treg cells, induced Treg cells, e.g. Tr1 and TH3 cells, as well as adaptive CD4+CD25high Treg cells developing in the periphery by conversion of CD4+CD25− T cells. All these different T cell populations with regulatory function coexist and contribute to immune suppression (Mills and McGuirk, Semin. Immunol.; 16: 107-117 (2004); Sakaguchi, Annu. Rev. Immunol.; 22: 531-562 (2004); Sakaguchi, Nat. Immunol.; 6: 345-352 (2005); Vigouroux et al., Blood; 104: 26-33 (2004)).
In the mouse CD25 is a good marker for Treg cells as animals are held under pathogen-free conditions. However, humans are constantly exposed to foreign antigens leading to a significant fraction of recently activated CD25+ effector T cells. In search of more specific Treg cell markers, the transcription factor FOXP3 has been identified as uniquely expressed in Treg cells in the mouse (Fontenot et al., Nat. Immunol.; 4: 330-336 (2003); Hori et al., Science; 299: 1057-1061 (2003); Khattri et al., Nat. Immunol.; 4: 337-342 (2003)) and expression has been proposed as a lineage marker already in developing Treg cells (Bennett et al., Nat. Genet.; 27: 20-21 (2001); Brunkow et al., Nat. Genet.; 27: 68-73 (2001)).
However, caution about its specificity still is recommended as recent reports in humans demonstrated induction of FOXP3 in activated conventional T cells without suppressive activity (Allan et al., J. Clin. Invest.; 115: 3276-3284 (2005); Morgan et al., Hum Immunol; 66: 13-20 (2005); Walker et al., J Clin Invest; 112: 1437-1443 (2003)). Recently developed mouse models suggest that FOXP3 is mandatory for the suppressive function of Treg cells and the final establishment of a Treg cell phenotype while lineage commitment apparently is independent of FOXP3 expression (Lin et al., Nat. Immunol.; 8: 359-368 (2007); Wan and Flayell, Nature; 445: 766-770 (2007); Williams and Rudensky, Nat Immunol; 8: 277-284 (2007)).
Characteristics of CD4+CD25highFOXP3+ Treg cells are their anergic state, their ability to actively inhibit CD4+CD25− T cells, CD8+ T cells, DC, NK, NKT, and B cells in a cell-cell contact and dose-dependent manner (Azuma et al., Cancer Res.; 63: 4516-4520 (2003); Chen, Front Biosci; 11: 1360-1370 (2006); Lim et al., J. Immunol.; 175: 4180-4183 (2005); Murakami et al., Proc. Natl. Acad. Sci. USA; 99: 8832-8837 (2002); Romagnani et al., Eur. J. Immunol.; 35: 2452-2458 (2005); Trzonkowski et al., Clin. Immunol.; 112: 258-267 (2004)). Phenotypically CD4+CD25highFOXP3+ Treg cells are characterized as antigen-experienced memory T cells, although lately some reports have described naïve CD4+CD25highFOXP3+ T cells in mice as well as humans (Beyer et al., Blood (2006); Valmori et al., J. Clin. Invest.; 115: 1953-1962 (2005)). Amongst the cell-surface markers associated with Treg cell phenotype and function cytotoxic T lymphocyte-associated protein 4 (CTLA4) and glucocorticoid-induced TNFR-related protein (GITR) are the most prominent molecules (McHugh et al., Immunity; 16: 311-323 (2002); Read et al., J. Exp. Med.; 192: 295-302 (2000); Shimizu et al., Nat. Immunol.; 3: 135-142 (2002); Takahashi et al., J. Exp. Med.; 192: 303-310 (2000)). Additionally, IL10 and TGFβ although rarely expressed in vitro might have functional importance for Treg cells in vivo, particularly in context of disease (Hara et al., J. Immunol.; 166: 3789-3796 (2001); Nakamura et al., J. Exp. Med.; 194: 629-644 (2001)). Major topics of current research are the characterization of Treg cell defects in autoimmune diseases and their role in infectious diseases and transplantation tolerance, particularly after allogeneic bone-marrow transplantation (Belkaid and Rouse, Nat. Immunol.; 6: 353-360 (2005); Hoffmann et al., Curr. Top Microbiol. Immunol.; 293: 265-285 (2005); Paust and Cantor, Immunol. Rev.; 204: 195-207 (2005); Sakaguchi, Annu Rev Immunol; 22: 531-562 (2004); Sakaguchi, Nat. Immunol.; 6: 345-352 (2005); Waldmann et al., Semin. Immunol.; 16: 119-126 (2004)). While research in Treg cell biology is intensifying, it is still unclear whether Treg cells in humans particularly in context of human diseases are mainly primed in the thymus or emerge in the periphery due to antigen-specific stimulation. The lack of more specific cell-surface markers is a major reason, why many functionally relevant aspects of Treg cells are still unknown.
Treg cells protect the host from autoimmune disease by suppressing self-reactive cells. As such, Treg cells may also block anti-tumor immune responses. Particularly in the context of cancer, Treg cell frequencies and function are important as increased numbers might favor tumor development or growth and influence the course of the disease. Currently a number of important questions are under intense investigation. Is the increase of Treg cell frequencies an early event at the onset of disease or more likely a response of the immune system during tumor progression? Do organ-specific and more important, do tumor-specific Treg cells play a role? How does therapy influence Treg cell numbers, particularly in already established tumors? Is there a possibility of long-term depletion of Treg cells and is this connected to induction of autoimmunity?
A number of mouse models could establish the development of Treg cells during tumor progression (Peng et al., J. Immunol.; 169: 4811-4821 (2002)). Relatively early induction of Treg cells during tumor development has significant impact in human disease as the time point of Treg cell induction in cancer patients certainly precedes the time of diagnosis in the majority of patients. Furthermore, a suppressive effect of naturally occurring Treg cells against tumor-specific CD8+ T cells was established in a poorly immunogenic B16 melanoma model (Turk et al., J. Exp. Med.; 200: 771-782 (2004)). Further evidence for the interference of Treg cells with CD8+ T cell-mediated anti-tumor immune responses in vivo was established in a transgenic murine colon carcinoma model where Treg cells abrogated CD8+ T cell-mediated tumor rejection by specifically suppressing cytotoxicity of CTL (Chen et al., Proc. Natl. Acad. Sci. USA; 102: 419-424 (2005)).
It has been suggested that suppression of anti-tumor immunity by Treg cells occurs predominantly at the tumor site and that local reversal of suppression, even late during tumor development, can be an effective treatment (Yu et al., J. Exp. Med.; 201: 779-791 (2005)).
Analysis of tumor-draining LN demonstrated that both anti-tumor effector T cells and FOXP3+ Treg cells are primed in the same LN during tumor progression. These tumor-antigen specific Treg cells possessed the same functional properties as Treg cells that arise naturally in the thymus (Hiura et al., J. Immunol.; 175: 5058-5066 (2005)).
Already before the identification of CD4+CD25+ Treg cells early data indicated that non-specific depletion of CD4+ T cells can lead to the induction of efficient anti-tumor immunity (Fu et al., Int. J. Cancer; 87: 680-687 (2000)). More specifically targeting Treg cells by administration of CD25 mAb abrogated immunological unresponsiveness to tumors and induced spontaneous development of tumor-specific CD8+ effector T cells and NK cells (Shimizu et al., J. Immunol.; 163: 5211-5218 (1999)). Interestingly, depletion of Treg cells led to cross-reactive tumor immunity against tumors of diverse origins (Golgher et al., Eur. J. Immunol; 32: 3267-3275 (2002)). Timing of Treg cell elimination also seems to be an important aspect. Administration of CD25 mAb later than 2 days after inoculation of myeloma cells caused no tumor regression, irrespective of Treg cell depletion (Onizuka et al., Cancer Res; 59: 3128-3133; (1999)). As already outlined this might be due to the induction of anti-tumor tolerance at a relatively early time-point of tumor development resulting in inefficient activation of effector cells. Furthermore the number of Treg cells after CD25 depletion is restored over time and the capacity to mount an anti-tumor response progressively diminishes (Casares et al., J. Immunol.; 171: 5931-5939 (2003)).
Depletion of Treg cells together with other immunostimulatory approaches, e.g. CTLA4 blockade, has also been tested. Combination of Treg cell depletion and CTLA4 blockade was synergistic and resulted in maximum tumor rejection. The observed synergism indicates that both pathways represent two alternatives for suppression of auto-reactive T cells so that simultaneous intervention might be a promising concept for the induction of therapeutic anti-tumor immunity (Sutmuller et al., J. Exp. Med; 194: 823-832 (2001)). Since immune responses to malignant tumors often are weak and ineffective, solely depleting Treg cells might not always result in tumor regression. Approaches combining Treg cell depletion with other immunological interventions, e.g. transfer of activated T cells or DC-based vaccinations, therefore might be more beneficial (Prasad et al., J. Immunol.; 174: 90-98 (2005); Tanaka et al., J. Immunother; 25: 207-217 (2002); Van Meirvenne et al., Mol. Ther.; 12: 922-932 (2005)).
It has long been recognized that cyclophosphamide exerts an immunostimulatory effect (Greenberg et al., J. Exp. Med.; 154: 952-963 (1981)). Early data indicated that cyclophosphamide preferentially destroys CD4+ suppressor T cells causing immunologically mediated regression of immunogenic lymphomas in mice (Awwad and North, Cancer Res.; 49: 1649-1654 (1989)). In a rat colon cancer model administration of cyclophosphamide depleted Treg cells and delayed the outgrowth of tumors (Ghiringhelli et al., Eur. J. Immunol.; 34: 336-344 (2004)). Combining cyclophosphamide and immunotherapy even cured the mice, while both strategies applied alone had no curative effect (Ercolini et al., J. Exp. Med.; 201: 1591-1602 (2005); Ghiringhelli et al., Eur. J. Immunol.; 34: 336-344 (2004)). Low-dose cyclophosphamide not only decreases numbers of Treg cells but also leads to decreased function, enhanced apoptosis and decreased homeostatic proliferation (Lutsiak et al., Blood; 105: 2862-2868 (2005)). This suggests that cyclophosphamide might be successfully integrated into chemoimmunotherapy as recently shown by Dudley et al. in Science; 298: 850-854 (2002). The combination of adoptive transfer of ex vivo activated tumor-specific T cells to lymphopenic melanoma patients after chemotherapy with cyclophosphamide and fludarabine induced tumor regression in up to 50% of patients treated.
Since Treg cells are an important cellular mechanism suppressing auto-antigen specific conventional T cells from attacking self tissues, non-specific depletion of these cells might be a too crude approach to be used without leading to significant collateral damage. The fine balance between benefit and harm of manipulating Treg cells was elegantly demonstrated in the following experiment: transfer of a mixture of CD4+CD25− and CD4+CD25+ T cells prevented effective adoptive immunotherapy of established melanoma. In contrast, adoptive transfer of CD4+CD25− T cells together with tumor—as well as self-reactive CD8+ T cells into CD4+ T cell deficient hosts followed by vaccination induced both regression of established melanoma but also severe and undesired autoimmunity (Antony et al., J. Immunol.; 174: 2591-2601 (2005)). Similarly, depletion of Treg cells with CD25 mAb in a mammary gland tumor model resulted in tumor regression but significantly increased susceptibility to autoimmune thyroiditis. This in vivo priming to both tumor- and self-antigens attests to the presence of otherwise undetectable immune effectors which are under negative regulation and demonstrates that modulation of Treg cells is a powerful strategy in cancer therapy, but may also significantly increase autoimmune complications (Wei et al., Cancer Res.; 65: 8471-8478 (2005); Wei et al., Cancer Immunol. Immunother.; 53: 73-78 (2004)).
Human Regulatory T Cells in Cancer—Current Knowledge and Open Questions
Already in the early 1990s T cells with regulatory function were reported in cancer patients, however, these reports were not followed up until the identification of CD4+CD25+ Treg cells in the mid 1990s (Sakaguchi et al., J. Immunol.; 155: 1151-1164 (1995)). Since then, an increase of Treg cells in cancer patients has been reported by numerous investigators. In contrast to the murine system, definition of human Treg cells has been more difficult and assessment of the most specific marker, namely FOXP3 has not been performed in many of the early studies. While human CD4+CD25high T cells are most enriched for FOXP3+ T cells, there are still significant numbers of FOXP3+ cells within the CD4+CD25low T cell population. In absence of more specific cell-surface markers, it is not yet possible to study human FOXP3+ Treg cells irrespective of their CD25 expression. These limitations also explain why Treg cells in humans currently need to be characterized by a combination of FOXP3 and CD25 expression as well as analysis of inhibitory function of T cell populations enriched for FOXP3+ cells, mainly by sorting CD25high T cells.
Comparability of previous reports is further challenged by use of different antibodies to detect CD25 or different gating strategies when assessing CD25+/CD25high cells. Similarly, function of Treg cells has been assessed with numerous in vitro approaches making it rather difficult to compare results of different studies.
Woo et al. were the first to report increased percentages of CD4+CD25+ Treg cells in TIL in non-small cell lung cancer and ovarian cancer (Woo et al., Cancer Res.; 61: 4766-4772; (2001)). These Treg cells were shown to secret TGFβ providing first evidence that Treg cells contribute to immune dysfunction in cancer patients (Woo et al., Cancer Res; 61: 4766-4772; (2001)). Further characterization of these cells showed constitutive high-level expression of CTLA-4. More important, Treg cells mediated potent inhibition of T cell proliferation (Woo et al., J. Immunol.; 168: 4272-4276 (2002)). Supporting this initial report, a larger study concluded that prevalence of CD4+CD25+ Treg cells is increased not only in the tumor microenvironment of patients with invasive breast or pancreas cancers but also in PB, suggesting that the increase of Treg cells is a generalized phenomenon (Liyanage et al., J. Immunol.; 169: 2756-2761 (2002)).
In malignant melanoma an increase of functional CD4+CD25+ Treg cells was observed (Javia and Rosenberg, J. Immunother.; 26: 85-93 (2003)), which was further linked to increases in the serum level of H-Ferritin (Gray et al., Clin. Cancer Res.; 9: 2551-2559 (2003)).
In patients with gastrointestinal malignancies the relative increase of Treg cells might actually be explained by a significant reduction of CD4+CD25− T cells. Interestingly, in patients with gastric carcinoma poor prognosis and decreased survival rates were closely correlated with higher Treg cell frequencies (Ichihara et al., Clin. Cancer Res.; 9: 4404-4408 (2003); Sasada et al., Cancer; 98: 1089-1099 (2003)). After curative resections, previously elevated Treg cells numbers were significantly reduced. In contrast, prevalence of Treg cells increased again in patients relapsing after tumor resection (Kono et al., Cancer Immunol. Immunother.; 1-8 (2005)). These findings underline the close correlation of tumor growth and Treg cell frequencies.
Curiel et al. demonstrated that CD4+CD25+FOXP3+ Treg cells suppress tumor-specific T cell immunity in ovarian cancer, contribute to tumor growth, and accumulate during progression (Curiel et al., Nat. Med.; 10: 942-949 (2004)). Furthermore, increased frequencies of Treg cells were associated with a high death hazard ratio and reduced survival. Treg cells preferentially moved to and accumulated in tumors and ascites, but rarely entered draining LN in later cancer stages. Tumor cells and surrounding macrophages produced the chemokine CCL22, which mediated trafficking of Treg cells to the tumor via CCR4. This specific recruitment of Treg cells might represent a mechanism by which tumors may foster immune privilege.
For patients with squamous cell carcinoma of the head and neck a significantly elevated frequency of FOXP3+GITR+ Treg cells was shown (Schaefer et al., Br. J. Cancer; 92: 913-920 (2005)). These Treg cells were significantly more sensitive to apoptosis than non-Treg cells which might hint at a rapid turnover in the peripheral circulation (Schaefer et al., Br. J. Cancer; 92: 913-920 (2005)). How the higher sensitivity to apoptosis influences Treg cells frequencies however has not been addressed yet.
Increased numbers of Treg cells have also been reported in PB and TIL of patients with hepatocellular carcinoma (Ormandy et al., Cancer Res.; 65: 2457-2464 (2005)). While the increase of Treg cells seems to be a common theme in solid tumors, there are clear but yet unexplained differences between individual tumor entities. In a comparative study differences in Treg cell frequencies were shown for malignant pleural effusions from patients with mesothelioma compared to carcinomatous pleural effusions from non-small cell lung cancer or breast cancer patients (DeLong et al., Cancer Biol. Ther.; 4: 342-346 (2005)).
Overall, previous work has clearly established that Treg cells are increased in most human solid tumors. Furthermore, there seems to be a stage dependent increase of Treg cells with frequencies of Treg cells probably correlated to overall survival. However, little is known about the mechanisms leading to this increase. A first study by Wolf et al. might help us to understand the underlying molecular mechanisms (Wolf et al., Cancer Immunol. Immunother.; 1-11 (2005)). In this study it was shown that increased frequencies of Treg cells in PB of cancer patients are due to active proliferation rather than redistribution from other compartments (i.e. secondary lymphoid organs or bone marrow). This finding in combination with the proposed attraction of Treg cells to the tumor via CCL22/CCR4 and induction of Treg cells by PGE2 or H-Ferritin might be one possible mechanism responsible for expansion of Treg cells in cancer patients.
While the question of Treg cells in solid tumors sparked interest relatively early, studies addressing Treg cells in hematological malignancies have been conducted only recently.
In patients with B cell chronic lymphocytic leukemia (CLL) a stage dependent increase of CD4+CD25highFOXP3+CTLA4+GITR+ Treg cells with full suppressive capacity could be established. However, when CLL patients were treated with fludarabine frequencies of Treg cells decreased and Treg cells showed impaired function. Ongoing studies are addressing the question how fludarabine mediates this effect (Beyer et al., Blood; 106: 2018-2025 (2005)). The increase of CTLA4+ Treg cells in untreated CLL patients which correlated with advanced disease stage and unfavorable cytogenetics was recently confirmed by others (Motta et al., Leukemia; 19: 1788-1793 (2005)). Similarly, in patients with B cell non-Hodgkin's lymphomas (B-NHL) increased frequencies of FOXP3+CTLA4+ Treg cells have been observed. PD1 expression was partly responsible for the suppressive activity of these LN infiltrating Treg cells. Furthermore, as reported for ovarian cancer, the tumor cells released CCL22 and thereby attracted CCR4+ Treg cells into the area of the lymphoma (Yang et al., Blood (2006)). For patients with acute myeloid leukemia (AML) higher frequencies of CD4+CD25high Treg have been observed. Similar to observations in solid tumors, Treg cells of AML patients were less resistant to apoptosis but showed higher proliferation compared to healthy individuals (Wang et al., Eur. J. Haematol.; 75: 468-476 (2005)).
Comparable to other hematological malignancies, increased frequencies of CD4+CD25highFOXP3+ Treg cells in patients with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) could be demonstrated (Beyer et al., Blood (2006)). Independent of prior therapy or stage of disease Treg cells exhibited a strong inhibitory capacity. Moreover, the increase of Treg cells was also stage-dependent and resulted from peripheral expansion. Furthermore, an expansion of naïve CD4+CD25highFOXP3+ Treg cells co-expressing CD45RA and CCR7 could be established for the first time further supporting the concept of peripheral expansion of this T cell compartment. The importance of identifying more specific markers as well as more standardized functional assays is supported by a recent report on FOXP3+ cells in PB from MM patients (Prabhala et al., Blood; 107: 301-304 (2006)). Due to an alternative experimental approach only assessing FOXP3 in context of CD4+ T cells but not CD25+ cells these data are difficult to compare with other studies on naturally occurring CD4+CD25high Treg cells. While this report came to the conclusion that Treg cells are dysfunctional in MM patients, the assays chosen to assess Treg cell function allowed for alternative explanations of the observed results including already described defects in conventional autologous T cells in MM patients (Mariani et al., Br. J. Haematol.; 113: 1051-1059 (2001)). To reconcile these recent findings about Treg cells it is most important to identify specific cell-surface markers for Treg cells that allow us to isolate these cells and functionally test them in context of malignant disease.
Taken together the concept of increased Treg cells has been established for solid tumors as well as hematological malignancies. However, more specific markers such as FOXP3 as well as more sophisticated and standardized functional assays have to be developed.
As already outlined a correlation of increased Treg cells with greater disease burden and poorer overall survival has been reported. In CLL reduced frequencies of functionally impaired Treg cells after fludarabine treatment could be observed, however not every chemotherapeutic agent seems to induce this effect as in CLL or MM no other treatment including autologous stem cell transplantation did induce similar effects. In line with this observation, frequency and suppressive function of Treg cells in tumor-draining LN derived from cervical cancer patients were not influenced by chemotherapy or combined chemoradiation (Fattorossi et al., Cancer; 100: 1418-1428 (2004)).
Recent work has demonstrated that IL2 signaling is required for thymic development, peripheral expansion and suppressive activity of Treg cells (Malek and Bayer, Nat. Rev. Immunol.; 4: 665-674 (2004)). During immune reconstitution after chemotherapy IL2 therapy led to a homeostatic peripheral expansion of Treg cells and to a markedly increased Treg cell compartment. IL2 therapy induced expansion of existent Treg cells in normal hosts and this expansion was further augmented by lymphopenia. Treg cells generated by IL2 therapy expressed FOXP3 at levels observed in healthy individuals and these Treg cells also were of similar potency suggesting that IL2 and lymphopenia are modulators of Treg cell homeostasis (Zhang et al., Nat. Med.; 11: 1238-1243 (2005)). Similarly, in patients with melanoma or renal cell carcinoma (RCC) the frequency of fully functional Treg cells was significantly increased after IL2 treatment, which was also accompanied by an increase of FOXP3 demonstrating that administration of high dose IL2 increases the frequency of circulating FOXP3+ Treg cells (Ahmadzadeh and Rosenberg, Blood (2005)). This might also explain why therapy with IL2 in RCC patients has not yet fully lived up to expectations since significant induction of Treg cells might counteract potential anti-tumor effects of IL2.
Surprisingly, vaccination of melanoma patients with DC either loaded with synthetic peptides or tumor lysates was also shown to induce increased frequencies of Treg cells, concomitant with the expansion of tumor-specific CTL. Whether this enhances anti-tumor tolerance and negatively influences the induction of clinically efficient anti-tumor immune responses needs further attention, since the mechanisms of this phenomenon are not yet understood (Chakraborty et al., Hum. Immunol.; 65: 794-802 (2004)).
Murine models have established that selective elimination of Treg cells alone or in combination with other treatment options might induce regression of already established tumors. First pilot studies have been initiated in cancer patients to selectively eliminate Treg cells. A promising and specific approach might be targeting of CD25 on the surface of Treg cells. Danull et al. used IL2 diphtheria toxin conjugate DAB(389)IL2 (denileukin diftitox, ONTAK) to selectively eliminate CD25-expressing Treg cells from the PBMC of cancer patients without inducing toxicity on other cells that only expressed CD25 at intermediate to low levels (Dannull et al., J. Clin. Invest.; 115: 3623-3633 (2005)). DAB(389)IL2 significantly reduced the number of Treg cells present in the PB of metastatic RCC patients and abrogated Treg cell mediated immunosuppressive activity in vivo. Moreover, elimination of Treg cells followed by vaccination with RNA-transfected DC significantly improved stimulation of tumor-specific T cell responses when compared with vaccination alone.
In summary, this first clinical study specifically eliminating Treg cells has shown promising results that need to be further evaluated. An important aspect of future studies will be to clearly describe the therapeutic window of deleting Treg cells as a major gate-keeper of self-antigen recognition.
In humans characterization of Treg cells has mainly focused on co-expression of CD4 and CD25 while differentiation status, frequencies of Treg cell subtypes, e.g. natural or induced Treg cells, Tr1 or TH3 cells are less well characterized. To better understand and study Treg cell biology in relation to tumor development and progression, it is most critical to identify more specific cell-surface markers as assessment of FOXP3 does not allow for subsequent functional testing of FOXP3+ cells.
Using whole genome transcription analysis 43 candidate genes were identified, which might serve as diagnostic as well as therapeutic targets for future clinical studies. Among these candidate genes six transcripts were further specified that were up to now only described as putatively expressed protein products and that were found specific for human Treg cells.